Peptide from soluble form of acetylcholinesterase, active as a calcium channel modulator

ABSTRACT

The acetylcholinesterase 14-mer peptide AEFHRWSSYMVHWK acts alone or in synergism with  2 -amyloid to contribute to neuronal degeneration, e.g. in Parkinson&#39;s and Alzheimer&#39;s diseases, perhaps by exerting calcium channel opening activity. Antibodies and other compounds which inhibit the biological activity are useful for prophylaxis or treatment.

[0001] This invention concerns the enzyme acetylcholinesterase (AChE) inwhich the inventors have identified a biologically active peptide.

[0002] The classical or cholinergic role of AChE is to degradeenzymatically extracellular acetylcholine. However, it has long beenknown that AChE exists also in a soluble form, (not a requirement forits classic enzymatic role) and is found in parts of the body wherethere is little or no acetylcholine. It is becoming widely accepted thatAChE has a non-cholinergic function, though the biochemical basis forthis function remains unclear.

[0003] It is believed that excessive AChE may enhance calcium entry intocells independent of its normal enzymatic action. Elevated cellularcalcium levels may lead to a range of pernicious consequences, includingundesirable changes in gene expression and, more importantly,mitochondrial swelling which may thereby compromise ATP metabolism andmay indeed lead to apoptosis or programmed cell-death. Disease stateswhich may be implicated include Parkinson's disease, Alzheimer'sdisease, stroke and malignancy.

[0004] The cytoplasm of cells typically contains calcium atconcentrations of the order of 1 μm. Calcium is present intracellularlyin the endoplasmic reticulum in millimolar concentrations. Extracellularbody fluids contain calcium also in millimolar concentrations. A calciumpump operates to maintain this substantial concentration differencebetween the cytoplasm and the endoplasmic reticulum, and thapsigargin isknown to be implicated in the breakdown of this pump. Similarly acalcium pump normally functions between the cytoplasm and theextracellular fluid. It is believed that the consequences of the actionof excessive AChE may be comparable to the breakdown of these pumps.

[0005] AChE, acting in a non-cholinergic capacity, has been shown toplay an important part in the normal and abnormal functioning of thesubstantia nigra, the region affected in Parkinson's disease. There areare three possible ways in which AChE may have toxic effects:

[0006] (i) excessive AChE may be released as a consequence ofcompensatory mechanisms known to occur in that disorder;

[0007] (ii) excessive glutamatergic activity known to occur inParkinson's disease may lead to over-stimulation of calcium channelN-methyl-D-aspartate (NMDA) glutamate receptors, thereby converting aphysiological situation to a pathological one;

[0008] (iii) normal levels of AChE may act synergistically withfragments of β-amyloid precursor proteins known to be present in theParkinsonian substantia nigra.

[0009] AChE, again acting in a non-cholinergic capacity, may be animportant contributing factor in Alzheimer's disease. In transgenic micewith excessive AChE there are cognitive deficits reminiscent ofAlzheimer's disease. Moreover Alzheimer's disease has been directlyassociated with inappropriate levels and forms of AChE. Excessive AChEmay act to enhance calcium entry through overactivation of otherwisenormal adaptive processes via a mechanism discussed in the experimentalsection below.

[0010] Current therapies for both degenerative diseases are somewhatinadequate. Anti-Parkinsonian drugs which target dopamine substitutiondo not arrest neuronal cell loss, and newer drugs aiming to blockcalcium entry directly may have poor net payoff in terms of neuronalhealth and in addition would have widespread undesirable effects in boththe central nervous system and peripheral tissue. Moreover, drugs usedin Alzheimer's disease which exclusively target the cholinergic system,neglect areas where AChE may be having its pivotal non-cholinergicfunction. Previous attempts to target calcium channel activity intherapy for neurodegenerative disorders have been hampered by thenon-selective effects of the compounds available.

[0011] In order to be conveniently administered, a compound fortreatment of disorders of the central nervous system, or moreparticularly of the brain, needs to be capable of crossing theblood-brain barrier. AChE is not capable of doing this, though a smalllipid-soluble analogue of part of this molecule might be. Workers in thefield have been seeking biologically active peptides based on the AChEmolecule for more than ten years, in the hope of thereby achieving amore effective and selective treatment for disorders of the centralnervous system such as Alzheimer's and Parkinson's diseases.

[0012] It is known that antagonism of NMDA receptors is being exploredas a therapy for stroke. The present invention is expected to findapplication in specific therapies for combating stroke and otherproblems of cerebral circulation.

[0013] Abnormal cholinesterase expression occurs in several types oftumour cells. Although the role of cholinesterases in tumorigenesis isunclear, the fact that AChE and BuChE (butyryl cholinesterase) may beinvolved in the control of cell growth and proliferation during earlydevelopment suggests that the amplification of cholinesterase genes mayinfluence the ability of tumour cells to proliferate more rapidly.According to the invention, antagonists of the non-cholinergic action ofAChE are expected to be of interest in the prophylaxis and treatment ofcancer.

[0014] Several separate lines of evidence suggest that motor neuronesmay share, along with the neurones that are lost in Parkinson's disease(substantial nigra) and in Alzheimer's disease (basal forebrain, locuscouruleus, raphe nucleus) several distinctive features as well as thecommon characteristic of releasing AChE in a non-cholinergic capacity.The released AChE may have a novel action, as in the regions prone toParkinsonian or Alzheimer degeneration to enhance developmentalmechanisms in immature populations of motor neurones but exert toxicactions if inappropriately reactivated in mature systems. TheAChE-peptide described herein may also therefore be pivotal in theaetiology of Motor Neurone Disease. The undisclosed finding supportingthis claim is that in pilot studies, the AChE peptide binds bilaterallyto selective sites within the spinal cord.

[0015] Amyloid precursor protein (APP) is known to have similar featuresto AChE as follows. Both AChE and APP are secreted from neurons into thecerebro spinal fluid (CSF), where for both AChE and APP there is adecrease in CSF levels in Alzheimer's disease. Both AChE and APP canhave trophic functions.

[0016] Both AChE and β-amyloid enhance calcium entry through NMDAreceptors. Both AChE and APP activate potassium channels, probablylinked to changes in intracellular calcium. Both AChE and β-amyloidactivate macrophages. Low stimulation of NMDA receptors has trophiceffects whereas high stimulation is toxic. The dual trophic-toxic actionof both APP and AChE may thus be mediated via NMDA receptors. A similardual action via NMDA receptors has already been shown for the trophicfactor BDNF in cortical cells. Finally, β-amyloid and the monomer ofAChE can bind together as a complex.

[0017] This invention results from the inventors' identification of aregion of the AChE molecule from which a biologically active peptide(obtained either synthetically or by endogenous processing) can bederived. The peptide consists of 14 residues of AChE from residue 535 toresidue 548 of the mature protein (in the translation of the mRNAsequence, EMBL accession hsache. empri, number M55040, beginning atnucleotide 310). The sequence of this peptide is amino Ala-Glu-Phe-His-Arg-Trp-Ser-Ser-Tyr-Met-Val-His-Trp-Lys-carboxy, or in the one lettercode, AEFHRWSSYMVHWK. The inventors propose that this, or a related,peptide from this region of AChE acts alone or in synergism with afragment of beta-amyloid to contribute to neuronal degeneration. Theinvention thus provides in one aspect a peptide containing at least sixamino acid residues and having at least 70% homology with part or all ofthe above sequence. Preferably the peptide contains at least 12 aminoacid residues having at least 90% homology with the above sequence.

[0018] It appears that the two amino acid residues-Val-His-, appearingat positions 11 and 12 in the above sequence, may be of criticalimportance. Thus the invention also envisages peptides comprising orconsisting of the four-mer sequence YMVH or MVHW or VHWK and having atleast 70% homology with part or all of the above AChE sequence.

[0019] A somewhat similar peptide is present in a region of theβ-amyloid precursor polypeptide. This region lies at the amino terminusof the 42 residue peptide that accumulates in Alzheimer's disease andhas the sequence amino-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-carboxy, or in the one letter code,DAEFRHDSGYEVHHQK, corresponding to residues 597-612 of the translationof the human amyloid A4 precursor polypeptide (EMBL accession hsafpa4.empri number Y00264, beginning at nucleotide 148).

[0020] The accompanying table shows the multiple sequence alignment of 5AChE sequences, three BuChE sequences and the human amyloid precursorpolypeptide at the region of interest. As reported in the experimentalsection below, the human amyloid precursor fragment does not itselfexert calcium channel opening activity, but it does enhance the activityof the AChE fragment. The BuChE fragment appears inactive, both aloneand together with the AChE fragment. In another aspect the inventionthus envisages a mixture of AChE peptide with another peptide having atleast four amino acid residues preferably including VH and having atleast 70% homology with the above β-amyloid precursor sequence.

[0021] There are various ways in which this AChE peptide or this peptidemixture may be used:

[0022] a) Since the AChE peptide is shown to have nanomolar affinity fora binding site in the vulnerable cells, the peptide (or mixture) can belabelled with a signal moiety, or alternatively immobilised, and used tolocate and identify the receptor site of the cells. The nature of thissignal moiety is not material, and the technique of labelling peptideswith signal moieties is well known. The peptide (or mixture) can be usedas an affinity ligand for the selective retrieval of the receptormolecule itself from preparations derived from those vulnerable cells.Additionally, the peptide affinity ligand could be used to screen anappropriate cDNA expression library to isolate a cDNA encoding thebinding site directly. Once that receptor site is known, it will bepossible to modify or control its properties.

[0023] b) An alternative and preferred approach is to find a substancethat inhibits the action of the biologically active peptide or mixture.For example an antibody or other substance which binds to the peptidewould be expected to inhibit its biological action. Structuralproperties of the active peptide itself, together with a combinatorialanalysis of the optimal peptide sequence for biological activity, willprovide additional information. This structural information may suggesta family of synthetic (non-peptide) compounds that could rationally betested for efficacy.

[0024] In a further aspect, the invention thus envisages a compoundwhich inhibits a biological activity of the AChE peptide or peptidemixture described above. The biological activity may perhaps bemodulating, directly or indirectly a calcium-channel-opening activity.The compound will preferably be capable of crossing the blood-brainbarrier.

[0025] Thus the non-cholinergic action of AChE, as mimicked by its 14residue peptide, may be selectively blocked by a synthetic compounddevised in this way. Moreover, the process of developing such asynthetic inhibitor is simplified by the demonstration of biologicalactivity in such a small sub-fragment of the AChE molecule. Aconsequence should be that the synthetic compound offers a morephysiological action, thus reduction of calcium entry into vulnerablecells rather than complete abolition. In addition, this action shouldoccur selectively, only in locations within the brain where AChE has anon-cholinergic action. It should be noted that these are the very sitesprimarily affected by cell loss in Alzheimer's and Parkinson's diseases.Thus use of these synthetic compounds should avoid widespread disruptionof cellular calcium regulation, by offering a highly region-selectiveaction within the brain.

EXAMPLE 1

[0026] Strategy for the Identification of a Receptor for the AChEPeptide

[0027] 1. Use the peptide, tagged with biotin, to search for a cell typewith a high affinity binding site for the peptide. Note that this searchwill begin with neuronal-derived tissue culture cell lines that shouldbe good candidates. Functional significance for the binding of the AChEpeptide will be assessed by looking for physiological effects of peptidebinding, such as transient calcium currents.

[0028] 2. Having identified a cell type with a high affinity bindingsite for the peptide, the receptor will be identified by ligand overlayblotting and intracellular localisation by indirect detection of thebiotinylated peptide using a streptavidin conjugated fluorochrome.Subsequently the receptor will be purified either by affinitychromatography using immobilised peptide, or by conventional columnchromatography using the ability of the peptide to bind to columnfractions as an assay to follow purification.

[0029] 3. The purified receptor will be subject to N-terminalmicrosequencing (or tryptic fragments will be purified by HPLC andmicrosequenced if the receptor molecule as isolated proves to beN-terminally blocked). The peptide sequences obtained in this way willbe compared with a non-redundant compilation of available peptidesequence databases to identify any similarities (or identity with aknown surface molecule). The sequences will also be compared withexpressed sequence tag (EST) databases in case the mRNA for the receptorhas already been obtained as a cDNA by chance in a random libraryconstruction and sequencing project.

[0030] 4. If the strategy in (3) does not identify a cDNA sequence, thepeptide sequences will be back-translated to provide nucleotidesequences from which oligonucleotides will be constructed. Theseoligonucleotides will be used to amplify regions of the parent mRNA byreverse transcription of total cellular RNA (from the cell type used inthe original biochemical isolation), followed by specific amplificationwith each possible primer pair using the polymerase chain reaction(PCR). These PCR products will be directly sequenced by cycle sequencingusing an Applied Biosystems automated sequencer.

[0031] 5. The sequences obtained from these PCR products will becompared with sequences in existing nucleotide databases as in. (3). Ifthis comparison reveals that an identical sequence has previously beenobtained, then a strong candidate for the receptor gene is available(Note that this does not imply that the sequence that has been obtainedpreviously has already been implicated in any way in the functions thatthe invention ascribes to this molecule).

[0032] 6. If no identical or highly similar sequences are identified in(5), then the PCR-derived nucleotide fragments will be used asradiolabelled probes to screen a cDNA library constructed by oligo-dTprimed reverse transcription from the mRNA of the cell type used in theoriginal biochemical isolation. This will identify candidate cDNA cloneswhich will then be sequenced as above. The identity of the candidatecDNAs with the protein of interest will be confirmed initially bydemonstrating that the clone contains the sequences of the otherPCR-derived nucleotide fragments. If an incomplete cDNA clone isobtained then 5′ extension will be carried out using the RACE technique(Rapid amplification of cDNA ends).

[0033] 7. The function of the protein encoded by the cDNA will beconfirmed by expression of the full-length protein using a transienteukaryotic expression vector in cells previously shown not to have ahigh affinity binding site for the AChE peptide. Expression of theprotein in these cells should result in the appearance of a highaffinity binding site for the AChE peptide on these transfected cells.This will confirm that the correct sequence has been identified.

[0034] 8. The cDNA sequence will be used to express the protein in abaculovirus-infected insect cell system in order to obtain large amountsof pure protein for structural studies. Note that this may require theconstruction of a soluble ectodomain fragment if the protein is atransmembrane molecule. The structural studies will include circulardichroism (CD) measurements, 2-D nuclear magnetic resonance analysis(NMR) and attempts at crystallisation. Pure receptor protein will alsofacilitate detailed analysis of the binding of peptides, or candidatenon-peptide agonists (or antagonists), to the receptor using surfaceplasmon resonance methods.

[0035] 9. Once the AChE ligand and its receptor are known, there arevarious ways of controlling or preventing their action:

[0036] a) Destroy the ligand, i.e. identify a protease that cleaves theactive peptide ligand into an inactive form and promote its activitye.g. by inducing it or impregnating it.

[0037] b) Prevent production of the ligand, i.e. identify a proteasethat produces the ligand and inhibit that.

[0038] c) Sequester the ligand and remove it with antibody or withsoluble receptor ectodomain. A modification of this approach is possibleif the AChE/β-amyloid synergy results from competition for a highaffinity sequestration site different from that which produces thebiological effect. Introduction of excess of high affinity site willreduce the biological effect.

[0039] d) Block the receptor with an antagonist, e.g. design an analoguethat binds the receptor, competes with endogenous peptide, but does notraise calcium levels. In an experimental system showing binding andcalcium ion signals, it will be possible to assay for a class ofcompounds that bind the receptor, compete with the peptide ligand, butdo not themselves activate the receptor.

[0040] e) Uncouple the receptor from the cellular response, e.g. bypreventing ligand binding to the receptor from causing cellular responseby blocking a second messenger that is preferably unique to the system.

EXAMPLE 2

[0041] The 14-mer AChE peptide, the corresponding 14-mer human BuChEpeptide (AGFHRWNNYMMDWK) and the 16-mer β-amyloid peptide weresynthesised and used in studies to assess their biological activity,with the summarised results given in Examples 3 to 7

[0042] Evidence for Alpha Helical Structure of the AChE 14-mer Peptide

[0043] The inventors inferred from the known alpha helical structure ofthe region of the beta amyloid peptide homologous to the AChE 14-merpeptide that the peptide was likely to adopt a helical conformation.This is significant because it is only in this helical conformation thatthe residues conserved between the acetylcholinesterase (AChE) sequenceand the amyloid precursor protein (APP) sequence are brought together toform a patch on one side of the peptide; it was suggested that thisstriking conservation exists because this patch is the interactionsurface with a second component, probably a cell surface receptor.Far-UV circular dichroism spectra have been obtained. The solvent usedis 95% trifluoroethanol; this is a standard condition for thedetermination of conformation in small peptides, and is the conditionused for published determination of β-amyloid peptide structure. Theresults clearly show that the AChE 14-mer peptide adopts a helicalconformation under conditions in which a related peptide derived fromthe homologous region of APP is also helical. These experiments alsoshow that the peptide from the homologous region ofbutyrylcholinesterase (BuChE) adopts a random coil configuration underthese conditions. This is significant since BuChE is regarded as anegative control because this enzyme lacks the non-cholinergic trophiceffects of AChE.

EXAMPLE 3

[0044] (i) Electrophysiological Studies

[0045] These experiments are performed on slices of guinea-pig midbrainmaintained in vitro. Intracellular recordings are made from a rostralpopulation of neurons in the substantia nigra under current clampconditions. All results listed below are obtained in the presence of thesodium channel blocker tetrodotoxin. In order to facilitatevisualisation of calcium-mediated potentials triggered by activation ofNMDA receptors, all experiments are performed in magnesium-freeperfusate. Under these conditions, results to date indicate:

[0046] (a) In 11 neurons, in concentrations ranging from 10⁻⁷M to 10⁻⁶M,the peptide fragment derived from AChE has a selective and reversibleaction reminiscent of the actions of AChE itself, i.e. with lowerdoses/less sensitive situations there is an enhanced calcium influx.This effect is followed by, in sustained applications/strongerdoses/more sensitive neurons, a marked reduction in the calciumpotentials.

[0047] (b) Under conditions where AChE is normally effective and undermagnesium-free conditions, the comparable BuChE fragment appears withoutcorresponding effect (n=3), and the analogous fragment of β-amyloid alsoappears ineffective (n=4). However, a synergism between the peptidederived from AChE and this fragment of β-amyloid is reflected in areduction in the evoked calcium potential (n=7) followed by thegeneration of large spontaneous thapsigargin-sensitive calcium currentsoscillating in a biphasic manner (n=3).

[0048] (c) In 6 neurons, application of NMDA, which on its own producesa ‘physiological’ depolarisation, results, under identical conditions,in severe metabolic stress of the cell after treatment with the AChEpeptide at a concentration as low as 10⁻⁷M, or at an even lowerconcentration when combined with the amyloid peptide.

[0049] High doses of NMDA, repeated electrical stimulation and indeedraised extra cellular calcium levels, all result in an effect on calciumpotentials similar to that seen for AChE peptide. The most obviouscommon factor in these three other treatments is that all to themenhance calcium entry; the most parsimonious explanation for thereduction in calcium potential, seen following AChE peptide, is that thepeptide enhances calcium entry too.

[0050] These results suggest that the peptide specified in the inventionis enhancing calcium entry into a population of neurons in thesubstantia nigra. Once large amounts of calcium have entered the neuron,buffering mechanisms come into play, reflected by the marked reductionin calcium potential. At its most effective, when the peptide iscombined with the fragment from β-amyloid, then this enhanced calciumentry followed by the triggering of intracellular control mechanisms, isseen as a spontaneous oscillation. It has already been shown thatrecombinant AChE, acting in a non-classical fashion, can enhance calciumentry into these neurons via a modulatory action on the NMDA receptor.These results suggest that the peptide derived from AChE, specified inthe invention, could be responsible for this effect.

[0051] (ii) Behavioural Studies

[0052] In these experiments, rats were chronically implanted with acannula in one substantia nigra and left to recover. After a period ofabout 3 days, they were infused with either a saline control solution,or a solution containing the 14-mer AChE peptide of the invention at adose of 1 μl of 10⁻⁵M. After a single infusion, they were challengeddaily with a systemic application of amphetamine for the subsequent 10days. Although the control group (n=6) showed no significant effects,the group receiving the peptide (n=8) gradually started to displaycontraversive rotation, which reached a maximum after 7 days postinfusion and remained consistent for the remaining 3 days tested.

[0053] These results suggest that the peptide-mediated calcium entryobserved in (i) could be setting in train long-latency, long-termintracellular events that result in a sustained elevation of theactivity of neurons in the treated substantia nigra. This enhanced,unilateral activation is manifest as contraversive circling behaviour.

[0054] AChE when infused unilaterally into the substantia nigra producesa long-term increase in circling behaviour which reflects increasedactivity of the nigrostriatal pathway. Under some circumstance thiseffect is mimicked by AChE-peptide, although the onset of the effecttakes several days following peptide infusion and the response isvariable. A low concentration of APP-peptide also increases activity ofthe nigrostriatal pathway, although doubling the concentration reversesthis effect, possibly reflecting the change from trophic to toxicactions of this agent. AChE- and APP-peptide appear to interact.

EXAMPLE 4

[0055] Electrophysiological Evidence for an Effect of the AChE 14-merPeptide on Neurones of the Hippocampus

[0056] The hippocampus is a brain region remote from the substantianigra detailed in the original application. This issue is importantbecause an effect of the peptide in the substantia nigra can beconnected to Parkinson's disease because cells in the substantia nigraare lost during the development of this condition. By contrast an effectin the hippocampus can be connected to Alzheimer's disease because thehippocampus is a major site of degenerative neuropathology inAlzheimer's disease.

[0057] Data has been obtained to suggest that the peptide has directtoxic effects on hippocampal cells in organotypic cultures. The effectis synergistic with the known excito-toxic effects ofN-methyl-D-aspartate (NMDA), can be seen within one hour of application.Toxic effects are also detectable histochemically over a culture periodof three weeks. This is particularly important because there is a needfor a demonstration of peptide toxicity in a system related to a majorneurodegenerative disorder.

[0058] The organotype tissue culture technique requires postnatal (day5-7) rats given terminal anaesthesia followed by decapitation. Sectionsof hippocampus, 400 μm thick, are prepared and then plated on aplasma/thrombin clot. A serum-based media is added to these cultures,which can then be maintained at constant temperature (35° C.) for up to21 days. After each study, cultures are stained with trypan blue toassess cell viability, in addition, after every removal of serum media,cultures are assayed for lactate dehydrogenase (LDH—a solublecytoplasmic enzyme used as an index of cellular damage).

[0059] Semi-acute application of the AChE-peptide and/orN-methy-D-aspartate (NMDA) for 1 hour results in extensive cellulardamage in various regions of hippocampal sections, compared to controlsamples.

[0060] Following chronic studies (cultures are maintained for 21 daysand treated with AChE-peptide and/or NMDA every 34 days), cultured cellsare immunocytochemically stained for acetylcholinesterase in order toassess what action, if any, the AChE-peptide has on cell number. Thefindings of biochemical studies (LDH assays) carried out on thesechronic cultures support the proposed toxic action of AChE-peptidefollowing its semi-acute application to hippocampal sections. However,current work suggests that acetylcholinesterase-positive cells may beprotected to some extent and, therefore it is possible that eitheracetylcholinesterase-negative cells are selectively vulnerable or theAChE-peptide may have a more pronounced action when applied with NMDA orboth these cases may apply.

EXAMPLE 5

[0061] Reproducible Binding of Peptide to Brain Sections

[0062] In order to obtain a probe derived from the AChE-peptide thatcould be followed when bound to specific sites within sections of brain(whether rodent or human) a modified peptide was made. The modifiedpeptide consisted of the AChE-peptide to which was covalently attachedat the N terminus a fluorescein group. This modified peptide was boundto fixed permeabilised brain sections, the excess unbound materialwashed off and then the fluorescein group detected with an alkalinephosphatase conjugated monoclonal anti-fluorescein reagent. This in turnwas washed to remove the unbound excess, and a substrate that produces ahighly localised, coloured, insoluble precipitate in the presence ofalkaline phosphatase was added in a solution of pH 8.4. Finally, thereaction was stopped by lowering the pH to neutral (pH 7.0) and thedistribution of the reaction product was examined by microscopy andphotography.

[0063] This indirect ligand overlay method provides a significantamplification of the weak signal due to peptide binding and overcomes ageneral problem of background due to non-specific sticking of first orsecond reagents. A control with the modified peptide omitted was alwaysincluded, and a drug, levamisole, was routinely used to block theactivity of alkaline phosphatase enzymes that are present naturally inthe tissue. The result is a clean assay that shows peptide-dependentbinding reactions confined to certain brain regions and certainpopulations of cells within those regions.

[0064] The specific example of the cerebellum is a good one; the peptidespecifically labels a population of cells with neuronal morphologywithin the granular cell layer of the cerebellum in rats, guinea pigsand humans. The cells labelled represent a subpopulation of the cellspresent in this highly cellular layer. In rodents the staining ispresent in cell bodies, but excluded from the nucleus, and extends intolong processes, some of which extend into the molecular cell layer,forming a net around the Purkinje cells, which are themselves negative.

EXAMPLE 6

[0065] AChE 14-mer Peptide

[0066] Rabbits were immunised by conventional protocols with adjuvantcontaining a modified AChE-peptide. In this case, the modification wasto construct a covalent cluster of four copies of the AChE-peptideconnected via three lysine residues. This structure is known as amulti-antennary peptide, or MAP-peptide, and is known to give rise tomore potent stimulation of the recipient's immune response in manycases. The rabbits were repeatedly immunised and test bleeds were usedto follow the development of a response to the antigen.

[0067] The resulting antiserum is a high titre polycolonal antiserumwith marked specificity for the AChE-peptide; although the MAP-AChEpeptide is recognised with the highest affinity, the reagent is still apotent for binding monomeric AChE-peptide alone. The anti-AChEreactivity is of the IgG subclass, indicating that a secondary responsehas occurred in the animals.

[0068] A skilled reader knows that a similar immunisation protocol inmice (giving the MAP-peptide in an adjuvant into the peritoneal cavityon a number of occasions) would give rise to an immune response to theAChE peptide in mice. This immune response could be immortalised byfusion of immunocompetent cells from the immunised mice (conventionallythe spleen but in principle lymph node also) with a nonsecreting myelomacell line using fusogens such as polyethylene glycol or electricaldischarges. Immortalised cell lines producing a reactivity of interestare derived clonally by screening assays based upon the solid phasebinding assay used to study the binding of the polyclonal antibody tothe peptide. The cells of interest are subcloned to purity, so theirproduct is a single immunoglobulin species and the resulting purehybridoma population is preserved by freezing in liquid nitrogen andused to produce the monoclonal antibody that is then used in all of theways that a skilled reader knows of for such reagents.

[0069] It is routinely possible to obtain the sequence of the variableregions of the immunoglobulins produced by hybridomas selected in thisway. This would be done (as in previously published protocols for otherunrelated monoclonal antibodies) by using the polymerase chain reactionto amplify and clone the variable region sequences from messenger RNAisolated from the hybridomas of interest. These regions are then clonedback into a recombinant background that permits the engineering of theirassociation with specific detecting enzymes or prosthetic groups fordetection or purification. The recombinant protein is produced inbacteria, or in insect cells using the baculovirus expression system, orin mammalian cell lines.

EXAMPLE 7

[0070] Competition Assay

[0071] In order to be sure that the antibody recognition of peptidesoccurs without the possible distortion due to the binding of the peptideto a charged plastic surface, an assay was used in which the criticalinteraction is studied in solution. Briefly, a solid phase assay was setup using the AChE-peptide on a plastic surface in a small well and anamount of the antibody which will give a large signal of known size whenassayed in this solid phase assay. An aliquot of the antibody waspreincubated with potential competitors for binding to the AChE-peptidebefore presenting the resulting mixture to the peptide bound to thewell. If the competitor has bound to the anti-peptide antibody duringthe preincubation, then there will be less free antibody available tobind to the immobilised peptide in the well, and the signal thatrecorded in the solid phase assay will be reduced.

[0072] This assay was first validated by demonstrating that theAChE-peptide itself could compete in this assay in a dose-dependentmanner, and was then used to show that the antiserum generated isspecific for AChE-peptide. This latter conclusion is drawn from the factthat even excessive doses of the APP or BuChE peptide could not competefor the binding of antibody to immobilised AChE-peptide.

[0073] When human CSF was used as the competitor during thepreincubation, high levels of competition were recorded showing thathuman CSF contains a component that is structurally similar to theepitope(s) present in the AChE-peptide.

[0074] The Western blotting assay in which the proteins of the CSF areelectrophoretically separated according to size and then transferred toa nitrocellulose substrate addresses the question of the identity of thespecies in human CSF that is recognised by the anti-peptide antiserum.The array of proteins on the nitrocellulose sheet (or blot) is probedwith the anti-peptide antibody followed by a secondary antibodycovalently attached to alkaline phosphatase. When this assay isperformed the anti AChE-peptide antiserum is found to specificallydecorate a protein component at approximately 25,000 Dalton molecularmass. This is not the size of the AChE. The protein is present in allCSF samples from both normal and Alzheimer's disease patients.

[0075] When the complex antiserum is affinity purified using the 25,000Dalton protein as the ligand, the resulting antibody recognises the25,000 Dalton protein as expected, but also a second, apparently lessabundant, protein. This second reactivity has the interesting andpotentially important property that the size of the protein is smallerin Alzheimer disease patients than in normal controls. Although thenumber of samples so far looked at is small (3 AD and 3 normal), theeffect is completely consistent, so perhaps this is a reproducibledifference between the CSF of normal and AD patients. If this isconfirmed, the potential for a diagnostic test is clear.

[0076] Peptide Length

[0077] The inventors have so far only studied one AChE 14-mer peptidelength; the evidence that polypeptides of other sizes may contain thefunctional region is that the anti-peptide antiserum recognises aspecies of 25,000 Daltons (much larger than the c2,000 Daltons of theAChE-peptide). Furthermore recombinant AChE that contains the part ofthe protein encoded by exon 6 is an effective competitor in the abovecompetition assay, showing that the region is still recognised by theantibody when it is attached to the parent protein.

[0078] Thus, the peptide used may not be uniquely functional because ofits size; the functional structure is encoded within the 14-mer, and canbe detected by antibody when present in the context of a much largerpolypeptide.

1 15 1 14 PRT Artificial Sequence Description of Artificial SequencePEPTIDE 1 Ala Glu Phe His Arg Trp Ser Ser Tyr Met Val His Trp Lys 1 5 102 16 PRT Artificial Sequence Description of Artificial Sequence PEPTIDE2 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 1015 3 4 PRT Artificial Sequence Description of Artificial SequencePEPTIDE 3 Tyr Met Val His 1 4 4 PRT Artificial Sequence Description ofArtificial Sequence PEPTIDE 4 Met Val His Trp 1 5 4 PRT ArtificialSequence Description of Artificial Sequence PEPTIDE 5 Val His Trp Lys 16 44 PRT Artificial Sequence Description of Artificial SequencePOLYPEPTIDE 6 Leu Ser Ala Thr Asp Thr Leu Asp Glu Ala Glu Arg Gln TrpLys Ala 1 5 10 15 Glu Phe His Arg Trp Ser Ser Tyr Met Val His Trp LysAsn Gln Phe 20 25 30 Asp His Tyr Ser Lys Gln Asp Arg Cys Ser Asp Leu 3540 7 54 PRT Artificial Sequence Description of Artificial SequencePOLYPEPTIDE 7 Ala Phe Trp Asn Arg Phe Leu Pro Lys Leu Leu Ser Ala ThrAsp Thr 1 5 10 15 Leu Asp Glu Ala Glu Arg Gln Trp Lys Ala Glu Phe HisArg Trp Ser 20 25 30 Ser Tyr Met Val His Trp Lys Asn Gln Phe Asp His TyrSer Lys Gln 35 40 45 Asp Arg Cys Ser Asp Leu 50 8 44 PRT ArtificialSequence Description of Artificial Sequence POLYPEPTIDE 8 Leu Ser AlaThr Asp Thr Leu Asp Glu Ala Glu Arg Gln Trp Lys Ala 1 5 10 15 Glu PheHis Arg Trp Ser Ser Tyr Met Val His Trp Lys Asn Gln Phe 20 25 30 Asp HisTyr Ser Lys Gln Glu Arg Cys Ser Asp Leu 35 40 9 44 PRT ArtificialSequence Description of Artificial Sequence POLYPEPTIDE 9 Leu Ser AlaThr Asp Thr Leu Asp Glu Ala Glu Arg Gln Trp Lys Ala 1 5 10 15 Glu PheHis Arg Trp Ser Ser Tyr Met Val His Trp Lys Asn Gln Phe 20 25 30 Asp HisTyr Ser Lys Gln Glu Arg Cys Ser Asp Leu 35 40 10 53 PRT ArtificialSequence Description of Artificial Sequence POLYPEPTIDE 10 Phe Trp AsnArg Phe Leu Pro Lys Leu Leu Asn Ala Thr Asp Thr Leu 1 5 10 15 Asp GluAla Glu Arg Gln Trp Lys Ala Glu Phe His Arg Trp Ser Ser 20 25 30 Tyr MetVal His Trp Lys Asn Gln Phe Asp His Tyr Ser Lys Gln Asp 35 40 45 Arg CysSer Asp Leu 50 11 42 PRT Artificial Sequence Description of ArtificialSequence POLYPEPTIDE 11 Thr Gly Asn Ile Asp Glu Ala Glu Trp Glu Trp LysAla Gly Phe His 1 5 10 15 Arg Trp Asn Asn Tyr Met Asn Asp Trp Lys AsnGln Phe Asn Asp Tyr 20 25 30 Thr Ser Lys Lys Glu Ser Cys Val Gly Leu 3540 12 47 PRT Artificial Sequence Description of Artificial SequencePOLYPEPTIDE 12 Lys Val Leu Glu Met Thr Gly Asn Ile Asp Glu Ala Glu GlnGlu Trp 1 5 10 15 Lys Ala Gly Phe His Arg Trp Asn Asn Tyr Met Asn AlaTrp Lys Asn 20 25 30 Asn Phe Asn Asp Tyr Thr Ser Lys Lys Glu Arg Cys AlaGly Phe 35 40 45 13 43 PRT Artificial Sequence Description of ArtificialSequence POLYPEPTIDE 13 Met Thr Gly Asp Ile Asp Glu Thr Glu Gln Glu TrpLys Ala Gly Phe 1 5 10 15 His Arg Trp Ser Asn Tyr Met Asn Asp Trp GlnAsn Gln Phe Asn Asp 20 25 30 Tyr Thr Ser Lys Lys Glu Ser Cys Thr Ala Leu35 40 14 70 PRT Artificial Sequence Description of Artificial SequencePOLYPEPTIDE 14 Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu TyrLys Met 1 5 10 15 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val HisHis Gln Lys 20 25 30 Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys GlyAla Ile Ile 35 40 45 Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val IleVal Ile Thr 50 55 60 Leu Val Met Leu Lys Lys 65 70 15 14 PRT ArtificialSequence Description of Artificial Sequence POLYPEPTIDE 15 Ala Gly PheHis Arg Trp Asn Asn Tyr Met Met Asp Trp Lys 1 5 10

1. A peptide containing at least 6 amino acid residues and having atleast 70% homology with part or all of the sequence AEFHRWSSYMVHWK.
 2. Apeptide comprising or consisting of the sequence YMVH or MVHW or VHWKand having at least 70% homology with part or all of the sequenceAEFHRWSSYMVHWK.
 3. A mixture of the peptide of claim 1 or claim 2 withanother peptide having at least 4 amino acid residues and having atleast 70% homology with the β-amyloid precursor sequenceDAEFRHDSGYEVHHQK.
 4. A probe consisting of the peptide of claim 1 orclaim 2 or the mixture of claim 3, labelled with a signal moiety, orimmobilised on a support.
 5. A compound which competes with the peptideof claim 1 or claim 2 for binding to a receptor therefor and whichthereby inhibits the biological activity of the said peptide.
 6. Acompound as claimed in claim 5, wherein the biological activity ismodulating a calcium-channel-opening activity.
 7. A compound as claimedin claim 5 or claim 6, which is capable of crossing the blood-brainbarrier.
 8. An antibody to the peptide of claim 1 or claim
 2. 9. Anantibody as claimed in claim 8 which is of the IgG class.
 10. Anantibody fragment or chimeric or humanised antibody comprising variableregions of the antibody of claim 8 or claim
 9. 11. A method of preparinga composition for treatment of disorders of the central nervous systemor stroke or cancer, which method comprises bringing a compoundaccording to any one of claims 5 to 10 into a form for humanadministration.
 12. A method of preparing a composition for controllingcytoplasmic calcium ion concentration in vivo, which method comprisesbringing a compound according to any one of claims 5 to 10 into a formfor human administration.